Fuel and energy production have emerged as one of the great challenges of the 21st century, and solving these problems touches upon an arena of issues that range from security to poverty to the environment. New approaches to providing for the world's energy needs are required to address these mounting concerns.
Among forms of plant biomass, lignocellulosic biomass (“biomass”) is particularly well-suited for energy applications because of its large-scale availability, low cost, and environmentally benign production. In particular, many energy production and utilization cycles based on cellulosic biomass have very low greenhouse gas emissions on a life-cycle basis. The primary obstacle impeding the more widespread production of energy from biomass feedstocks is the general absence of low-cost technology for overcoming the recalcitrance of these materials to conversion into useful products. Lignocellulosic biomass contains carbohydrate fractions (e.g., cellulose and hemicellulose) that can be converted into ethanol or other end-products including lactic acid and acetic acid. In order to convert these fractions, the cellulose or hemicellulose must ultimately be converted or hydrolyzed into monosaccharides. This hydrolysis has historically proven to be problematic.
Cellulose digesting anaerobic bacteria are of great potential utility because they can be used to produce ethanol or other fuels from abundant substrates such as forestry, municipal and agricultural waste. However, it has been challenging to realize this potential utility because of difficulty in the genetic manipulation of these organisms and lack of understanding of their metabolic biochemistry. Genome sequence data and recent advances in biotechnological tools for genetic modification of Clostridium thermocellum and other similar organisms have made it possible to make progress in this area, but the great complexity of metabolism makes it difficult to achieve efficiently a desired outcome such as near theoretical ethanol yield from cellulosic substrates.
Many microorganisms can metabolize glucose, cellulose or cellodextrins anaerobically, but they vary in the pathways utilized and the products generated. It has been demonstrated in genetically modified Thermoanaerobacterium saccharolyticum that glucose and cellobiose can be fermented to ethanol at very close to theoretical yield, but similar genetic manipulations in Clostridium thermocellum have not had the same outcome. Argyros, D A, Tripathi S A, Barrett T F, Rogers S R, Feinberg L F, Olson D G, Foden J M, Miller B B, Lynd L R, Hogsett D A, Caiazza N C, High ethanol titers from cellulose using metabolically engineered thermophilic, anaerobic microbes. Appl. Env. Microbiol. 2011. 77(23):8288-94.
Clostridium thermocellum has both cellulolytic and ethanologenic fermentation capabilities and can directly convert a cellulose-based substrate into ethanol. However, C. thermocellum possesses a branched carbon utilization pathway that generates products other than ethanol and is not as amenable to manipulation for ethanol production as that of T. saccharolyticum. This is exemplified more clearly when the carbon utilization pathways from the two organisms are compared. In homoethanologenic T. saccharolyticum, the carbon atoms from glucose flow down a linear central metabolic pathway to ethanol (FIG. 1A). In C. thermocellum, a different set of enzymes is present and thus the carbon utilization pathway (FIG. 1B) is different than in T. saccharolyticum. This difference in the carbon-utilization pathways in these organisms makes it infeasible to produce ethanol at theoretical yield with the same modifications.
The invention relates to cellulose-digesting organisms that have been genetically modified to allow the production of ethanol at a high yield by redirecting carbon flux at key steps of central metabolism. Redirection means altering the flux of carbon from the normally prevailing routes to alternate routes by means of genetic modification.